*Target (protein/gene name): MtnX Phospatase *NCBI Gene # or RefSeq#: 30264114 1088838 *Protein ID (NP or XP #) or Wolbachia#: NP_846491.1 *Organism (including strain): Bacillus anthracis str. Ames
Bacteria; Firmicutes; Bacilli; Bacillales; Bacillaceae; Bacillus; Bacillus cereus group. Etiologic Risk Group (see link below): Appendix B-II-A. Risk Group 2 (RG2) - Bacterial Agents *Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Bacillus anthracis is the cause or origin of the disease commonly known as anthrax. B.anthracis is a rod shaped bacterium, that forms endospores and is gram-positive. It can be grown in aerobic or anaerobic conditions and is one of the few bacterium's that is known to synthesize the protein capsule, poly-Y-D-gamma-glutamic acid.This acid capsule of Bacillus anthracis is the lethal toxin agent found in anthrax [1]. The PGA capsule is able to avoid immune cell regulation and can proliferate within the host, making it an important factor in the pathogenesis of anthrax infection. MtnX plays an important role in the function of this organism because it catalyzes the dephosphorylation of 2-hydroxy-3-keto-5-methylthinopentenyl-1-phosphate into mtnD [2]. This MtnX phosphatase contributes to energy supply and regulation within Bacillus anthracis that is necessary for the bacterium to function.
FIgure 1. BRENDA enzyme mechanism schematic of MtnX Phosphatase methionine salvage pathway in Bacillus subtilis.
Information for the mechanism schematic could not be found for organism Bacillus anthracis on BRENDA. However, MtnX functions similarly in organism Bacillus subtilis.
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): spectrophotometic -- link to Sigma (or other company) page for assay or assay reagents (substrates): p-Nitrophenyl Phosphate Liquid Substrate System
pNPP does not work in Klebsiella pneumoniae version -- link (or citation) to paper that contains assay information: Crystal structure of MtnX phosphatase from Bacillus subtilis at 2.0 Å resolution provides a structural basis for bipartite phosphomonoester hydrolysis of 2-hydroxy-3-keto-5-methylthiopentenyl-1-phosphate -- List cost and quantity of substrate reagents and supplier:
N7653-100ML, $79.90 (Sigma-Aldrich, USA) Structure Available (PDB or Homology model) MtnX Phosphatase with Organism Bacillus anthracis is not available on the PDB webpage. However, MtnX Phosphatase with organism Bacillus subtilis is provided and the information for that is included below. It is recommend that a Homology model is created for this phosphatase. -- PDB # or closest PDB entry if using homology model:2FEA -- For Homology Model: Will be required.
Figure 2. Pairwise alignment of BLASTP search of MtnX Phosphatase in Bacillus anthracis in NCBI against MtnX Phosphatase in Bacillus subtilis in PDB.
---- Query Coverage: 97% ---- Max % Identities: 52% ---- % Positives:167/214(78%) ---- Chain used for homology FASTA: Protein Database (2FEA) for protein sequence MtnX in Bacillus subtilis that will be used for homology model:
>2FEA:A|PDBID|CHAIN|SEQUENCE
GMTTRKPFIICDFDGTITMNDNIINIMKTFAPPEWMALKDGVLSKTLSIKEGVGRMFGLLPSSLKEEITSFVLEDAKIRE
GFREFVAFINEHEIPFYVISGGMDFFVYPLLEGIVEKDRIYCNHASFDNDYIHIDWPHSCKGTCSNQCGCCKPSVIHELS
EPNQYIIMIGDSVTDVEAAKLSDLCFARDYLLNECREQNLNHLPYQDFYEIRKEIENVKEVQEWLQNKNAGESSLK NCBI Reference Sequence (NP_846491.1) protein sequence MtnX in Bacillus anthracis:
>gi|30264114|ref|NP_846491.1| 2-hydroxy-3-keto-5-methylthiopentenyl-1-phosphate phosphatase [Bacillus anthracis str. Ames] MSIQVFCDFDGTITNNDNIMSIMEKFAPPEAEEVKNRILSQELSIQEGVSQLFQLIPTNLHDEIIQFLIE TAEIRNGFHEFIQFVNENNISFYVISGGMDFFVYPLLQGLIPKEQIYCNETDFSNEYITVNWPHPCDRLC QNHCGLCKSSLIRKLSDTNDFHIVIGDSITDLQAAKQADKVFARDFLITKCEENHISYTPFETFHDVKTE LKHLLEVKL
Current Inhibitors: N/A Expression Information (has it been expressed in bacterial cells): Escherichia coli Purification Method: N/A Image of protein (PyMol with features delineated and shown separately):
Figure 3. Biological Assembly Image for 2FEA Crystal structure of MtnX phosphatase from Bacillus Subtilis at 2.00 A resolution in PyMOL.
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): MSIQVFCDFDGTITNNDNIMSIMEKFAPPEAEEVKNRILSQELSIQEGVSQLFQLIPTNLHDEIIQFLIE TAEIRNGFHEFIQFVNENNISFYVISGGMDFFVYPLLQGLIPKEQIYCNETDFSNEYITVNWPHPCDRLC QNHCGLCKSSLIRKLSDTNDFHIVIGDSITDLQAAKQADKVFARDFLITKCEENHISYTPFETFHDVKTE LKHLLEVKL *length of your protein in Amino Acids: 219aa Molecular Weight of your protein in kiloDaltons using the Expasy ProtParamwebsite: 25289.7 kDa Molar Extinction coefficient of your protein at 280 nm wavelength:
Ext. coefficient 13325 Abs 0.1% (=1 g/l) 0.527, assuming all pairs of Cys residues form cystines (with Cys)
Ext. coefficient 12950 Abs 0.1% (=1 g/l) 0.512, assuming all Cys residues are reduced (without Cys) TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
Figure 4. TMpred output for 2-hydroxy-3-keto-5-methylthiopentenyl-1-phosphate phosphatase [Bacillus anthracis str. Ames]
*CDS Gene Sequence (paste as text only): ATGAAACGTATTAAAATTTCAACAGAGTATATTACACTAGGGCAATTTTTAAAGTTAGCCGATGTAATTG ATACAGGTGGCGCTGTAAAATGGTTTTTACAAGAATATGAAGTGTACGTGAATCAAGAACTTGAAAATAG AAGAGGGCGCAAGCTATATGCGAACGATATTATTGAAATTCCAGGAAGCGGAAGTTTCCAAGTTCAGTCA TAA *GC% Content for gene: 34.74% *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): N/A (not needed currently) *GC% Content for gene (codon optimized): N/A (not needed Currently)
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to. Primer design results for 'tail' primers (this is just 2 sequences):
References:
[1] Jang J., The poly-y-D-glutamic acid capsule of Bacillus anthracis enhances lethal toxin activity. Epub2011, 79 (9), 3846-54.
[2] Xu Q., Crystal structure of MtnX phosphatase from Bacillus subtillis at 2.0 angstroms resolution provides a structural basis for bipartite phosphomonoester hydrolysis of 2-hydroxy-3-keto-5-methylthiopentenyl-1-phosphate. PubMed2007, 69 (2), 433-9.
*NCBI Gene # or RefSeq#: 30264114
1088838
*Protein ID (NP or XP #) or Wolbachia#: NP_846491.1
*Organism (including strain): Bacillus anthracis str. Ames
Bacteria; Firmicutes; Bacilli; Bacillales; Bacillaceae; Bacillus; Bacillus cereus group.
Etiologic Risk Group (see link below): Appendix B-II-A. Risk Group 2 (RG2) - Bacterial Agents
*Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Bacillus anthracis is the cause or origin of the disease commonly known as anthrax. B.anthracis is a rod shaped bacterium, that forms endospores and is gram-positive. It can be grown in aerobic or anaerobic conditions and is one of the few bacterium's that is known to synthesize the protein capsule, poly-Y-D-gamma-glutamic acid.This acid capsule of Bacillus anthracis is the lethal toxin agent found in anthrax [1]. The PGA capsule is able to avoid immune cell regulation and can proliferate within the host, making it an important factor in the pathogenesis of anthrax infection. MtnX plays an important role in the function of this organism because it catalyzes the dephosphorylation of 2-hydroxy-3-keto-5-methylthinopentenyl-1-phosphate into mtnD [2]. This MtnX phosphatase contributes to energy supply and regulation within Bacillus anthracis that is necessary for the bacterium to function.
Essentiality of this protein: Essential
http://onlinelibrary.wiley.com/doi/10.1002/prot.21602/full
Complex of proteins?: No
Druggable Target: Yes
*EC#: 3.1.3.87
Link to BRENDA EC# page: 3.1.3.87
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): spectrophotometic
-- link to Sigma (or other company) page for assay or assay reagents (substrates):
p-Nitrophenyl Phosphate Liquid Substrate System
pNPP does not work in Klebsiella pneumoniae version
-- link (or citation) to paper that contains assay information:
Crystal structure of MtnX phosphatase from Bacillus subtilis at 2.0 Å resolution provides a structural basis for bipartite phosphomonoester hydrolysis of 2-hydroxy-3-keto-5-methylthiopentenyl-1-phosphate
-- List cost and quantity of substrate reagents and supplier:
N7653-100ML, $79.90 (Sigma-Aldrich, USA)
Structure Available (PDB or Homology model)
MtnX Phosphatase with Organism Bacillus anthracis is not available on the PDB webpage. However, MtnX Phosphatase with organism Bacillus subtilis is provided and the information for that is included below. It is recommend that a Homology model is created for this phosphatase.
-- PDB # or closest PDB entry if using homology model: 2FEA
-- For Homology Model: Will be required.
---- Max % Identities: 52%
---- % Positives:167/214(78%)
---- Chain used for homology FASTA:
Protein Database (2FEA) for protein sequence MtnX in Bacillus subtilis that will be used for homology model:
>2FEA:A|PDBID|CHAIN|SEQUENCE
GMTTRKPFIICDFDGTITMNDNIINIMKTFAPPEWMALKDGVLSKTLSIKEGVGRMFGLLPSSLKEEITSFVLEDAKIRE
GFREFVAFINEHEIPFYVISGGMDFFVYPLLEGIVEKDRIYCNHASFDNDYIHIDWPHSCKGTCSNQCGCCKPSVIHELS
EPNQYIIMIGDSVTDVEAAKLSDLCFARDYLLNECREQNLNHLPYQDFYEIRKEIENVKEVQEWLQNKNAGESSLK
NCBI Reference Sequence (NP_846491.1) protein sequence MtnX in Bacillus anthracis:
>gi|30264114|ref|NP_846491.1| 2-hydroxy-3-keto-5-methylthiopentenyl-1-phosphate phosphatase [Bacillus anthracis str. Ames] MSIQVFCDFDGTITNNDNIMSIMEKFAPPEAEEVKNRILSQELSIQEGVSQLFQLIPTNLHDEIIQFLIE TAEIRNGFHEFIQFVNENNISFYVISGGMDFFVYPLLQGLIPKEQIYCNETDFSNEYITVNWPHPCDRLC QNHCGLCKSSLIRKLSDTNDFHIVIGDSITDLQAAKQADKVFARDFLITKCEENHISYTPFETFHDVKTE LKHLLEVKL
Current Inhibitors: N/A
Expression Information (has it been expressed in bacterial cells): Escherichia coli
Purification Method: N/A
Image of protein (PyMol with features delineated and shown separately):
MSIQVFCDFDGTITNNDNIMSIMEKFAPPEAEEVKNRILSQELSIQEGVSQLFQLIPTNLHDEIIQFLIE TAEIRNGFHEFIQFVNENNISFYVISGGMDFFVYPLLQGLIPKEQIYCNETDFSNEYITVNWPHPCDRLC QNHCGLCKSSLIRKLSDTNDFHIVIGDSITDLQAAKQADKVFARDFLITKCEENHISYTPFETFHDVKTE LKHLLEVKL
*length of your protein in Amino Acids: 219aa
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParamwebsite: 25289.7 kDa
Molar Extinction coefficient of your protein at 280 nm wavelength:
Ext. coefficient 13325 Abs 0.1% (=1 g/l) 0.527, assuming all pairs of Cys residues form cystines (with Cys)
Ext. coefficient 12950 Abs 0.1% (=1 g/l) 0.512, assuming all Cys residues are reduced (without Cys)
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
ATGAAACGTATTAAAATTTCAACAGAGTATATTACACTAGGGCAATTTTTAAAGTTAGCCGATGTAATTG ATACAGGTGGCGCTGTAAAATGGTTTTTACAAGAATATGAAGTGTACGTGAATCAAGAACTTGAAAATAG AAGAGGGCGCAAGCTATATGCGAACGATATTATTGAAATTCCAGGAAGCGGAAGTTTCCAAGTTCAGTCA TAA
*GC% Content for gene: 34.74%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): N/A (not needed currently)
*GC% Content for gene (codon optimized): N/A (not needed Currently)
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
References:
[1] Jang J., The poly-y-D-glutamic acid capsule of Bacillus anthracis enhances lethal toxin activity. Epub 2011, 79 (9), 3846-54.
[2] Xu Q., Crystal structure of MtnX phosphatase from Bacillus subtillis at 2.0 angstroms resolution provides a structural basis for bipartite phosphomonoester hydrolysis of 2-hydroxy-3-keto-5-methylthiopentenyl-1-phosphate. PubMed 2007, 69 (2), 433-9.